The ability of secretory and endocytic pathways to sort and distribute proteins to the appropriate compartments is essential to maintain the functional and structural organization of eukaryotic cells. The overall goal of this project is to understand the molecular basis of selectivity that governs specific transport steps in the endocytic and secretory pathways. Defects in these pathways are likely to contribute to the etiology of multigenic diseases such as cancer and heart disease. Analysis of clathrin-mediated protein transport in yeast suggests that clathrin participates in transport pathways from the Golgi and the plasma membrane that ultimately leads to the lysosome-like vacuole. During the previous funding period, insights were achieved that allow unique approaches to address three important transport steps in these pathways: 1) clathrin coated vesicle (ccv) uncoating; 2) cargo recognition during actin-based edocytosis; 3) receptor sorting in endosomes. A combination of genetic, biochemical, and cell biological strategies will be applied to address specific aims focused on each of these processes. First, the mechanism of ccv uncoating in vivo will be determined by characterization of Aux1p, a member of the DNA J-domain family of Hsc70 co-chaperones. Interactions between Aux1p and clathrin will be analyzed and effects of interaction mutants on ccv uncoating in vivo will be determined. The uncoating roles of Hsc70 family members will be evaluated and additional factors involved in uncoating will be sought by protein-interaction and genetic strategies. Second, the role of the actin-associated protein Sla1p in linking cargo to the actin-based endocytosis machinery will be characterized. Sla1p sequences involved in recognition of an ubiquitin-independent endocytic targeting signal will be mapped, mutated and mutants tested in vivo. The function of Sla1p domains in endocytosis will be characterized, and a role for phosphorylation in regulating Sla1p endocytosis activity will be investigated. Third, a potentially novel role for ubiquitin in sorting within endosomes will be addressed. The ubiquitin-dependent sorting step and the type of ubiquitin modification necessary for sorting will be defined. The role of cargo sequences in the sorting process will be analyzed. Ubiquitin-modified components of the sorting apparatus will be sought by protein interaction, genetic and biochemical strategies. Together these studies are expected to provide significant advances in understanding key aspects of intracellular protein transport: cargo selection, vesicle coat dynamics, and sorting within endosomes.